The Journal of Neuroscience, February 27, 2013 • 33(9):3953–3966 • 3953 Neurobiology of Disease ␣ Reducing GABAA 5 Receptor-Mediated Inhibition Rescues Functional and Neuromorphological Deficits in a Mouse Model of Down Syndrome Carmen Martínez-Cue´,1 Paula Martínez,1 Noemí Rueda,1 Rebeca Vidal,1,3,4 Susana García,1 Vero´nica Vidal,1 Andrea Corrales,1 Juan A. Montero,2 A´ngel Pazos,1,3,4 Jesu´s Flo´rez,1 Rodolfo Gasser,5 Andrew W. Thomas,5 Michael Honer,5 Fre´de´ric Knoflach,5 Jose Luis Trejo,6 Joseph G. Wettstein,5 and Maria-Clemencia Herna´ndez5 Departments of 1Physiology and Pharmacology and 2Anatomy and Cellular Biology, Faculty of Medicine, University of Cantabria, E-39011 Santander, Spain, 3Institute of Biomedicine and Biotechnology (University of Cantabria–Spanish National Research Council–Cantabria Research, Development and Innovation), E-39011 Santander, Spain, 4Center for Biomedical Research Network on Mental Health, Health Institute Carlos III, E-28029 Madrid, Spain, 5F. Hoffmann-La Roche, Pharma Research and Early Development, Discovery and Translational Area Neuroscience, CH-4070 Basel, Switzerland, and 6Department of Molecular, Cellular, and Developmental Neurobiology, Cajal Institute, Spanish National Research Council, E-28002 Madrid, Spain Down syndrome (DS) is associated with neurological complications, including cognitive deficits that lead to impairment in intellectual functioning. Increased GABA-mediated inhibition has been proposed as a mechanism underlying deficient cognition in the Ts65Dn (TS) mousemodelofDS.WeshowthatchronictreatmentofthesemicewithRO4938581(3-bromo-10-(difluoromethyl)-9H-benzo͓f͔imidazo[1, ␣ 5-a][1,2,4]triazolo[1,5-d][1,4]diazepine), a selective GABAA 5 negative allosteric modulator (NAM), rescued their deficits in spatial learning and memory, hippocampal synaptic plasticity, and adult neurogenesis. We also show that RO4938581 normalized the high density of GABAergic synapse markers in the molecular layer of the hippocampus of TS mice. In addition, RO4938581 treatment sup- pressed the hyperactivity observed in TSmice without inducing anxiety or altering their motor abilities. These data demonstrate that reducing GABAergic inhibition with RO4938581 can reverse functional and neuromorphological deficits of TS mice by facilitating brain plasticity and ␣ support the potential therapeutic use of selective GABAA 5 NAMs to treat cognitive dysfunction in DS. Introduction including learning and memory deficits (Escorihuela et al., 1995; Down syndrome (DS) is the most common genetic cause of in- Reeves et al., 1995; Holtzman et al., 1996) with alterations in both tellectual disability (Bittles et al., 2007). The disorder is caused by hippocampal morphology (Insausti et al., 1998; Kurt et al., 2004) trisomy of chromosome 21 and is associated with neurological and adult neurogenesis (Rueda et al., 2005; Clark et al., 2006; complications, including cognitive deficits that lead to impair- Llorens-Martín et al., 2010). ment in intellectual functioning (Lott and Dierssen, 2010). There Excessive GABA-mediated neurotransmission has been pro- is currently no pharmacological therapeutic option available for posed as one of the underlying causes of the cognitive deficits in the treatment of cognitive deficits in people with DS. TS mice (Belichenko et al., 2004; Fernandez et al., 2007). TS mice The genetic dependence of the cognitive phenotype in DS has display fewer asymmetric synapses that mediate excitatory trans- been recapitulated in mouse models of the condition of which the mission in the temporal cortex and dentate gyrus (DG) (Kurt et Ts65Dn (TS) mouse is the most widely used (Kahlem et al., 2004). al., 2000, 2004) and synaptic structural abnormalities in the hip- This murine model shows several fundamental features of DS, pocampus and cortex, including a selective reorganization of the inhibitory input (Belichenko et al., 2004; Pe´rez-Cremades et al., 2010). In addition, enhanced GABA-mediated inhibition was as- Received March 9, 2012; revised Dec. 14, 2012; accepted Jan. 3, 2013. Author contributions: C.M.-C., A.P., J.F., F.K., J.L.T., J.G.W., and M.-C.H. designed research; P.M., N.R., R.V., S.G., sociated with deficient synaptic plasticity in the hippocampus of V.V.,A.C.,J.A.M.,R.G.,A.W.T.,andM.H.performedresearch;C.M.-C.,N.R.,R.V.,V.V.,F.K.,andM.C.H.analyzeddata; TS mice through a marked reduction in long-term potentiation C.M.-C. and M.C.H. wrote the paper. (LTP) in the CA1 and DG areas (Siarey et al., 1997; Kleschevnikov This work was supported by F. Hoffmann-La Roche, the Jerome Lejeune Foundation, and Spanish Ministry of et al., 2004; Costa and Grybko, 2005; Fernandez et al., 2007). EducationandScienceGrantsBFU2008-04397andBFU2011-24755.WethankE.GarcíaIglesias,M.Ca´rcamo,andR. Furthermore, overexpression of Olig1 and Olig2, two genes trip- Madureirafortheirtechnicalassistance,Drs.T.BallardandG.Trubeforhelpfuldiscussions,andS.Ortega,S.Gradari, and P. Pe´rez-Domper for advice on experiments. licated in DS and TS mice, are linked to the neurogenesis defects R.G., A.W.T., M.H., F.K., J.G.W., and M.-C.H. are employed by F. Hoffmann-La Roche. that led to an imbalance between excitatory and inhibitory neu- This article is freely available online through the J Neurosci Open Choice option. rons and to increased inhibitory drive in the TS forebrain Correspondence should be addressed to Dr. Maria-Clemencia Hernandez, Pharma Research and Early Develop- (Chakrabarti et al., 2010). In line with these findings, previous ment, Discovery and Translational Area Neuroscience, F. Hoffmann-La Roche, Grenzacherstrasse 124, CH-4070 Basel, Switzerland. E-mail: [email protected]. studies showed that reducing inhibitory neurotransmission by DOI:10.1523/JNEUROSCI.1203-12.2013 chronic administration of nonselective GABAA receptor antago- Copyright © 2013 the authors 0270-6474/13/333953-14$15.00/0 nists reversed the deficits in LTP and hippocampal-mediated 3954 • J. Neurosci., February 27, 2013 • 33(9):3953–3966 Martínez-Cue´ et al. • Enhancement of Brain Plasticity in Ts65Dn Mice memory of TS mice (Fernandez et al., 2007; Rueda et al., 2008a). How- cages. The dose of 20 mg/kg RO4938581 in 150 ␮l of chocolate milk was ever, nonselective GABA receptor antagonists are anxiogenic and pro- chosen because it led to plasma concentrations that correlated with 50– A ␣ convulsant (Dorow et al., 1983; Little et al., 1984). 70% GABAA 5 receptor occupancy under these conditions. During the ␣ behavioral assessment, drug was made available 1 h before testing; all Among the different GABAA receptor subtypes, GABAA 5 subunit-containing receptors are preferentially localized in the mice regularly consumed the entire chocolate milk–drug mixture. For the study of potential convulsant activity of RO4938581 in TS mice, the hippocampus and play a key modulatory role in cognition compound was prepared in 0.3% Tween 80 v/v 0.9% saline with 100 ml (Collinson et al., 2002; Crestani et al., 2002; Rudolph and Knoflach, of H O and administered orally by gavage (50 mg/kg) to achieve higher ␣ 2 2011). Moreover, selective GABAA 5 negative allosteric modu- plasma and brain exposures. lators (NAMs), also called inverse agonists, have cognition- enhancing effects without anxiogenic or proconvulsant side Behavioral studies effects (Collinson et al., 2006; Dawson et al., 2006; Nutt et al., The experimenters were blind to genotype and pharmacological treat- 2007; Ballard et al., 2009). Here we show that RO4938581 (3- ment throughout the entire behavioral assessment. Mice from cohort bromo-10-(difluoromethyl)-9H-benzo͓f͔imidazo[1,5-a][1,2,4] one were used for behavioral testing (actimetry, sensorimotor testing, ␣ triazolo[1,5-d][1,4]diazepine), a selective GABAA 5 NAM, can rotarod, and hole board tests). To decrease the chances of behavioral reverse concomitantly electrophysiological, neuromorphologi- responses being altered by previous test history, the most invasive pro- cal, and cognitive deficits of TS mice. cedures were performed last. Studies were performed in the following order: actimetry, sensorimotor testing, rotarod, open field, and hole Materials and Methods board. For cohort two, mice were assessed in the plus maze. Animals Spatial learning and memory: Morris water maze This study was approved by the Cantabria University Institutional Lab- To evaluate spatial learning and memory, a modified version of the Morris oratory Animal Care and Use Committee and performed in accordance water maze was used. The apparatus was a circular tank 110 cm in diameter, with the Declaration of Helsinki and the European Communities Council full of water (22–24°C) made opaque by the addition of powdered milk. Directive (86/609/EEC). For experiments performed at F. Hoffmann-La Inside the tank, a platform was hidden 1 cm below the water level. Roche, the experimental procedures received previous approval from the Mice were studied at the end of the drug treatment period in 16 con- City of Basel Cantonal Animal Protection Committee based on adher- secutive daily sessions: 12 acquisition sessions (platform submerged), ence to federal and local regulations on animal maintenance and testing. followed by a probe trial and four cued sessions (platform visible) (Fig. TS mice were generated by repeated backcrossing of B6EiC3Sn a/A- 1a). All trials were videotaped with a camera located 2 m above the water Ts(17Ͻ16Ͼ)65Dn females with C57BL/6Ei ϫ C3H/HeSNJ (B6EiCSn) level. The Anymaze computerized tracking system (Stoelting) was used F1 hybrid males. The